New drugs are needed for treatment of the disease human African trypanosomiasis (HAT) that is caused by the protist Trypanosoma brucei. Protein tyrosine (Tyr) phosphorylation is important for regulating numerous cellular processes in eukaryotes, and inhibition of Tyr phosphorylation by protein Tyr kinases (PTKs) has yielded several well-tolerated drugs that are in clinical use. In trypanosomes, Tyr phosphorylation is understudied, and the Tyr phosphorylation pathway has not been exploited to produce new lead drugs. Our long-term project goals are to (i) employ phenotypic assays to discover chemical scaffolds that inhibit Tyr phosphorylation of trypanosome proteins; (ii) optimize the scaffolds for pharmacokinetic, and physicochemical properties while preserving selectivity in trypanocidal activity over host cells; (iii) identify targets that bind the optimized leads; and (iv) evaluate te best-performing optimized analogs in acute and chronic murine models of HAT. Towards these goals, we have performed a focused chemical screen of drugs directed against human Tyr kinases, and identified 7 hits that killed axenic bloodstream T. brucei at low micromolar concentrations. Subsequently, we tested three of the drugs in a mouse model of HAT and found that the human Tyr kinase inhibitor lapatinib (GlaxoSmithKline) controls the trypanosome infection and cures 25% of mice infected with the parasite; we therefore deemed lapatinib to be a Lead compound. We initiated a lead optimization program that has produced 7 novel compounds with nanomolar activity in phenotypic assays against bloodstream T. brucei. We will pivot on our discovery of NEU617 which has an effective concentration of 42 nanomolar, to continue our optimization of lapatinib analogs to achieve better pharmacokinetic, physicochemical and improved selectivity and toxicity profiles. Using lapatinib as the prototype, we have developed multiple approaches for identifying the targets of these potent novel leads, and we will apply these techniques to identify targets of our optimized leads, and to chemically and genetically validate targets of the potential drugs. The best compounds from these optimization studies will be evaluated for efficacy in murine models of HAT.